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Citation: Es-sbata, I.; Castro, R.;
Carmona-Jiménez, Y.; Zouhair, R.;
Durán-Guerrero, E. Influence of
Different Bacteria Inocula and
Temperature Levels on the Chemical
Composition and Antioxidant
Activity of Prickly Pear Vinegar
Produced by Surface Culture. Foods
2022,11, 303. https://doi.org/
10.3390/foods11030303
Academic Editor: Charis M. Galanakis
Received: 18 December 2021
Accepted: 20 January 2022
Published: 24 January 2022
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4.0/).
foods
Article
Influence of Different Bacteria Inocula and Temperature Levels
on the Chemical Composition and Antioxidant Activity of
Prickly Pear Vinegar Produced by Surface Culture
Ikram Es-sbata 1,2 , Remedios Castro 1, Yolanda Carmona-Jiménez 1, Rachid Zouhair 2
and Enrique Durán-Guerrero 1, *
1Analytical Chemistry Department, Faculty of Sciences-IVAGRO, University of Cadiz, Agrifood Campus of
International Excellence (CeiA3), Polígono Río San Pedro, s/n, 11510 Puerto Real, Cadiz, Spain;
ikram.essbataess@alum.uca.es (I.E.-s.); remedios.castro@uca.es (R.C.); yolanda.carmona@uca.es (Y.C.-J.)
2Laboratory of Plant Biotechnology and Molecular Biology, Department of Biology, Faculty of Sciences,
Moulay Ismail University, Meknes 11201, Morocco; r.zouhair@umi.ac.ma
*Correspondence: enrique.duranguerrero@uca.es; Tel.: +34-956-016456
Abstract:
This work intends to determine the effect on the aroma profile, phenolic content and
antioxidant activity of prickly pear vinegars produced by the surface culture at two different fermen-
tation temperatures and using different acetic acid bacteria (AAB) inocula. Prickly pear wine was
fermented at two temperature levels (30 and 37
◦
C) by using bacteria inocula containing Acetobacter,
Gluconobacter or a mixture of bacteria isolated from Sherry vinegars. Eighty-five individual volatile
compounds from different families and sixteen polyphenolic compounds have been identified. It was
confirmed that the highest temperature tested (37
◦
C) resulted in a lower concentration of volatile
compounds, while no significant effect on the vinegars’ volatile composition could be associated with
the AAB inoculum used. Contrariwise, the highest content of polyphenolic compounds was detected
in those vinegars produced at 37
◦
C and their concentration was also affected by the type of AAB
inoculum used. Prickly pear wine displayed greater antioxidant activity than juices or vinegars, while
the vinegars obtained through the mixture of AAB from Sherry vinegar showed higher antiradical
activity than those obtained through either of the two AAB genera used in this study. It can be
therefore concluded that, although the volatile content of vinegars decreased when fermented at a
higher temperature, vinegars with a higher content in polyphenols could be obtained by means of
partial fermentations at 37 ◦C, as long as thermotolerant bacteria were employed.
Keywords:
prickly pear; vinegar; fermentation; thermotolerant bacteria; volatile compounds;
polyphenolic compounds
1. Introduction
Opuntia ficus-indica (L). Mill. is a plant that belongs to the Cactaceae family and can
grow in arid and semi-arid climates. Its common name is prickly pear, nopal cactus or cactus
pear. It originally comes from tropical or subtropical American regions, but it has already
been naturalized on all continents [
1
]. In Morocco, around one hundred and fifty thousand
hectares of area are cultivated with O. ficus-indica and the annual production of prickly
pear under optimal conditions can reach up to 20 tons dry matter/hectare/per year [
2
].
The prickly pear fruit pulp is considered to be the most edible and interesting part to be
processed for alimentary purposes. These fruits contain health-promoting substances, since
they are a good source of nutrients such as polyphenols and betalains [
3
]. The biochemical
composition of cactus fruits changes over their development and ripening. This depends
not only on the plant variety but also on the color, number of seeds, size, sugars, fats,
proteins, pectins and non-volatile organic acids contents [4].
Foods 2022,11, 303. https://doi.org/10.3390/foods11030303 https://www.mdpi.com/journal/foods
Foods 2022,11, 303 2 of 23
Vinegar is a food condiment that is worldwide produced from a variety of raw materi-
als, that can be vegetables, fruits or grains. Many types of vinegar are currently commer-
cialized, including balsamic, rice, wine or black vinegar. These vinegar varieties present
unique characteristics, flavors and tastes depending on the raw material used for their
production, the fermenting procedures and the microorganisms involved in the process [
5
].
Prickly pear juice, because of its high sugar content and low acid blend, seems to be
an interesting alternative for the production of vinegar [
2
]. Obtaining vinegar from cactus
pear juice would be a new way to valorize cactus fruit through a simple process that can be
performed at different industrial scales [
6
]. Two vinegar production methods are generally
recognized, the slow traditional or surface method and the quick industrial or submerged
one [
7
]. Both the method employed, as well as the fermentation time allowed, have an
impact on vinegar quality. Vinegars obtained through the traditional method usually
have a better sensory quality, with particularly richer aromas than those of the vinegars
elaborated using the industrial method. On the other hand, the submerged method is a
more economical process that leads to greater yields and is therefore generally preferred by
industrial producers [
8
,
9
]. Vinegar quality is the end result of a number of factors, such as
raw material, acetification conditions and acetic acid bacteria metabolism, which involves
the production of acetic acid from ethanol as well as other transformation processes such
as oxidations or the formations of esters, among others [10].
A number of studies have already revealed how important the production method
is with respect to the vinegar’s final aroma profile and hence to their organoleptic quali-
ties
[9,11]
. Beside the production method, there are other parameters that exert an influence
on the proportion of certain compounds, such as volatile and phenolic compounds, which
are key to determining vinegar quality—certain acetification parameters, such as the
amount of oxygen, the optimum temperature and the substrate loading, are some of these
key indicators [
12
]. In order to control and optimize vinegar quality, some recent studies on
fruit vinegar production have focused on the isolation of certain acetic acid bacteria, on the
processing conditions and on the vinegars’ phenolic and aromatic profiles [
13
]. Apart
from the above-mentioned parameters, vinegar production using different bacteria strains,
depending on their tolerance to acetic acid high concentrations, has been tested [14]; thus,
at lower concentrations of acetic acid in the medium, certain species of the Acetobacter genus
predominate, where Acetobacter aceti is generally the most often employed for vinegar pro-
duction [
15
]; however, when the acetic acid content reaches over 5%, the species from the
Gluconacetobacter genus are more effective [
14
,
16
,
17
]. Moreover, both high temperature and
high ethanol concentrations have an evident effect on vinegar fermentation processes; thus,
thermotolerant and ethanol-resistant strains are expected to become feasible technologies
for effective vinegar fermentation under unfavorable acetification conditions.
Each vinegar
'
s sensory characteristics, as well as its different flavors and aromas, are
determined by the pungent flavor of the acetic acid used for its production. Organic acids,
essential amino acids, vitamins, minerals, volatile compounds and other fermentation
products also play a major role regarding the organoleptic properties of vinegars [18].
Vinegar aroma, in particular, which depends on a large number of volatile compounds,
is one of its most determinant characteristics with regard to food quality and consumer
acceptance. These compounds may either be present in the raw material itself or may
be generated over the production process [
19
]. In the particular case of prickly pear
fruit vinegar, alcohols and esters are considered to be key aroma substances, responsible
for its characteristic faint melon or cucumber-like aroma. 2-(E/Z)-2,6-nonadien-1-ol and
2-methylbutanoic
acid methyl ester have been identified as the volatile compounds that
confer cactus fruit vinegar with their distinctive aroma [
20
]; however, in a recent study,
Farag et al. reported that the predominant volatile compounds in this fruit were short-chain
aldehydes (25–32%) and acids (25–29%) [21].
Phenolic compounds, on the other hand, seem to be secondary metabolites that are
closely related to the color and flavor traits of fruits, juices, and wines [
22
]. Some recent stud-
ies on Opuntia spp. stated that cactus pear fruit is a good candidate to develop new healthy
Foods 2022,11, 303 3 of 23
food, because of their high content of biofunctional polyphenolic compounds [
23
,
24
]; thus,
the polyphenolic fingerprint of prickly pear products is mainly characterized by the pres-
ence of flavonols and phenolic acids [
25
,
26
]. Particularly, among the minor compounds in
O. ficus-indica, betalains and polyphenols seem to be the most valuable antioxidants with
regard to the nutritional quality of prickly pears and of their transformation products [
27
].
Other studies have reported that prickly pear juice is rich in phenolic compounds that act
as effective radical scavengers [
28
]. Several polyphenolic compounds, mainly represented
by ferulic acid derivative, rutin, gallic acid and catechin, have been identified in prickly
pear pulp [
29
,
30
]. A simple way to evaluate the global antioxidant potential of a food,
due to the presence of the different bioactive substances present such as polyphenols, is the
determination of its antioxidant activity. Although there are clear differences between the
antioxidant activity
in vitro
and
in vivo
[
31
], the
in vitro
measurement could give an initial
idea of the possible healthy character of a certain food product.
In view of all the above said, this study intends to investigate and evaluate, on the
one hand, the effect of different fermentation temperatures and bacteria inocula on the
concentration of volatile and phenolic compounds in final prickly pear vinegars, and, on the
other, to determine the antioxidant activity of a number of prickly pear samples. In the
present study, three types of acetic acid bacteria inocula have been used to accomplish the
fermentation of a Moroccan prickly pear wine to produce prickly pear vinegar under two
temperatures (30 and 37
◦
C), all of them according to a surface culture production method.
To the best of our knowledge, this is the first work that deals with the analytical characteri-
zation of prickly pear vinegars produced under different surface culture conditions.
2. Materials and Methods
2.1. Fruit
The prickly pear fruits (yellow-orange color) from wild cultivars grown in Marrakesh-
Safi-Morocco region (coordinates: 31◦370N 8◦00W) were harvested during August (2019).
Each fruit piece was manually peeled off and the pulp was weighed and preserved under
frozen storage (
−
80
◦
C) until further procedures. The prickly pear fruit pulps were me-
chanically crushed using a conventional household blender. Then, the pulp mixture was
homogenized and filtrated using a colander (0.5 mm mesh size) in order to separate the
juice from the seeds. The juice was stored at 4
◦
C until its analysis. The
◦
Brix,
◦
Baumé, pH
and density values of the final juice were measured at 20
◦
C by means of a DMA 4500 M
densimeter (Anton Paar, Graz, Austria).
2.2. Alcoholic Fermentation
The alcoholic fermentation was performed at a pilot scale using stainless-steel tanks
covered by mosquito nets to avoid any contamination by insects. Twenty-five liters of
the final prickly pear juice were fermented in duplicate (50 L total volume) and the fer-
mentation temperature was maintained at a constant 22
◦
C to avoid any losses of volatile
compounds during the process. 60 mg/L of total sulfur dioxide (potassium metabisulphite,
Agrovin, Spain) was added to prevent the growth of undesirable microorganisms. A total of
0.35 g/L of diammonium phosphate was also added as a nutrient to the matrix; 0.20 g/L of
Saccharomyces cerevisiae active dry yeast, previously activated at 35
◦
C for 20 min were also
inoculated to start the fermentation process (Enartis Ferm SB, Trecate, Italy). The process
was monitored by measuring the sugar content in the substrate. After the fermentation
had been started, the sugar content was increased until 14
◦
Brix degrees was reached
(equal to the initial
◦
Brix value of the juice) by adding commercial white refined beet sugar
(AB Azucarera Iberia, Madrid, Spain) suitable for human intake, to rise the final alcoholic
degree. Then, the prickly pear wine produced was centrifuged at 15,000
×
gfor 10 min and
stored at 4 ◦C.
Foods 2022,11, 303 4 of 23
2.3. Acetic Fermentation
2.3.1. Bacterial Preparation
Ten thermo-ethanol tolerant strains (5 Acetobacter and 5 Gluconobacter; AAB) were
previously isolated from Moroccan prickly pear fruit following the procedure described
in the previous work [
32
]. Further, a mixture of strains from an unfiltered Sherry vinegar
(Jerez, Spain) was used in order to compare the different acetification profiles obtained
from different bacteria inocula. The selected AAB were precultured in a broth medium
and incubated at 30
◦
C for 24 h with continuous and vigorous agitation in order to initiate
the acetic acid bacterial cells' rapid proliferation. When the measured optical density (OD
600 nm) of the suspension reached over 1.2, the cells with 10% (v/v) of inoculum were
transferred into the prickly pear wine to start the acetic fermentation according to the
traditional method.
2.3.2. Surface Culture Acetification
The acetification of the prickly pear wine through surface culture was carried out in
500 mL Erlenmeyer flasks which had been previously sterilized and covered with cotton
sheets. The flasks were filled with 250 mL of prickly pear wine and each one of them was
then inoculated with 10% (v/v) of the different types of AAB inocula. The experiments
were conducted at 30
◦
C and 37
◦
C in duplicate and the flasks were not agitated to allow the
atmospheric oxygen to diffuse slowly into the fermenting medium. A temperature of 37
◦
C
showed the best results in terms of production of acetic acid by thermotolerant bacteria in
a previous work [
32
] and it was compared with the usual acetification temperature (30
◦
C).
The content in each flask was sampled every three weeks and each sample’s total acidity
was determined by titration with NaOH using phenolphthalein indicator. The total acidity
content was expressed as g of acetic acid/100 mL of vinegar. When the acidity content
stopped increasing, the fermentation process was considered as completed.
2.4. Analysis of Volatile Compounds
2.4.1. Sample Preparation
The samples were analyzed by SBSE/GC-MS according to the method described
by Durán Guerrero et al. [
19
]. The volatile compounds from the vinegar samples, were
extracted by means of commercially available polydimethylsiloxane stirs bars, 10 mm length
x 0.5 mm film thickness (supplied by Gerstel, Mülheim a/d Ruhr, Germany). For each stir
bar sorptive extraction (SBSE) analysis, a volume of 25 mL of the sample was pipetted and
placed into a 100-mL Erlenmeyer flask and 5.85 g of NaCl (Scharlau, Barcelona, Spain) was
added and dissolved by agitation. Then, 84
µ
L of 4-methyl-2-pentanol solution (Sigma,
Steinheim, Germany) (2.27 g/L in Milli-Q water containing 80 g/L of acetic acid) were also
added to the sample. The Erlenmeyer flasks were placed on a magnetic stirrer (Gerstel),
and they were stirred at 1250 rpm at 25
◦
C for 120 min. Then, the stir bars were removed
from the vinegar samples and placed for a few seconds in distilled water, in order to remove
the NaCl as well as any remains of prickly pear pulp that might have stuck onto them. They
were then gently dried using a lint-free tissue. Finally, the dried stir bars were transferred
into a glass thermal desorption tube and they were thermally desorpted.
2.4.2. Instrumentation
The thermal desorption system used for the coated stir bars was a commercial TDS-2
thermal desorption unit (Gerstel) connected to a programmed-temperature vaporization
(PTV) injector CIS-4 (Gerstel) through a heated transfer line. The PTV was installed onto
the chromatographic system. The thermo-desorption unit was equipped with an MPS
2L autosampler (Gerstel) with the capacity to handle 98 coated stir bars. The desorption
temperature was configured to go from 40
◦
C up to 300
◦
C (held for 10 min) at 60
◦
C/min
under a 75 mL/min helium flow and the desorbed analytes were cryofocused using the
PTV system with liquid nitrogen at
−
140
◦
C. Finally, the PTV system was configured to go
from
−
140
◦
C up to 300
◦
C (held for 5 min) at 10
◦
C/s and the analytes were analyzed by
Foods 2022,11, 303 5 of 23
Gas Chromatography–Mass Spectrometry (GC-MS). An Agilent 6890 GC-5973N MS system
(Agilent, Little Falls, DE, USA), equipped with a DB-Wax capillary column (J&W Scientific,
Folsom, CA, USA), 60 m
×
0.25 mm i.d. with a 0.25
µ
m coating, which was used on the
electron impact mode to perform the capillary GC–MS analysis. A 1.0 mL/min helium
flow was used as the carrier gas. The peaks were identified by means of the Wiley library
according to their mass spectra analogies (>85% matching) and confirmed by the retention
times of the standards, when available, or by the retention data found in the literature.
The linear retention index of each one of the compounds was determined by means of
a DB-Wax polar column and compared against those reported in the literature [
33
–
35
].
The semi-quantitative data were obtained by measuring the relative base of the ion peak
area in relation to that of 4-methyl-2-pentanol, as the internal standard. All the analyses
were performed in duplicate.
2.5. Analysis of Phenolic Compounds
The phenolic compounds in the prickly pear samples were identified and quantified by
means of a Waters Acquity UPLC system (Waters Corps. Milford, MA, USA), equipped with
a diode array detector (DAD) and following the method proposed by Schwarz et al. [
36
].
An Acquity UPLC BEH C18 column (100
×
2.1 mm/ID, with 1.7
µ
m particle size), also
from Waters, was used. All the samples (juice, wine and vinegars) were previously filtered
through 0.22 µm nylon filters manufactured by Scharlab (Barcelona, Spain).
The phenolic compounds were identified by comparing retention times and ultraviolet-
visible (UV-VIS) spectra against those of their corresponding commercial standards (Fluka,
Buchs, Switzerland; Sigma, Steinheim, Germany; and East Kodak, Rochester, NY, USA).
Each identified compound was quantified by comparison against the calibration curve
obtained from their corresponding standard at 280 nm (for gallic acid, hydroxy-tyrosol,
epigallocatechin, catechin, tyrosol, vanillic acid, syringic acid, ethyl gallate, m-coumaric
acid, hesperidin and naringenin), 320 nm (for protocatechualdehyde, p-coumaric acid,
ferulic acid, quercetin and cinnamic acid) and 255 nm (for p-hydroxybenzoic acid) at seven
concentration levels, except for hydroxy-tyrosol, which was quantified as tyrosol. All the
analyses were conducted in duplicate.
2.6. Analysis of the Antioxidant Activity
The antioxidant activity levels of the prickly pear juice, the wine and the different vine-
gars were determined by DPPH (1,1-diphenyl-2-picrylhydrazyl) according to the method
reported by Carmona-Jiménez et al. [
37
]. A total of 200
µ
L of sample or ethanol (blank) were
added into vials containing 3.3 mL of a 50
µ
M solution of DPPH in ethanol prepared daily
(0.069 ppm of the initial DPPH). Then, the mixture was allowed to sit at room temperature
for 3 h, the absorbance at 515 nm was measured using a Cary 50 Bio spectrophotometer (Var-
ian, Australia). All the measurements were conducted in duplicate. The exact concentration
(ppm) of the DPPH solution in the different samples was calculated spectrophotometrically
based on a calibration curve that was determined by linear regression:
y = 0.0284[DPPH] −0.011, R2= 0.9997 (1)
The inhibition percentage of DPPH of each sample at the steady state was determined
according to the following equation:
I (%) = [(Abs blank −Abs sample)/Abs blank] ×100 (2)
2.7. Statistical Analysis
An analysis of variance (ANOVA) with Tukey’s test was used to initially determine
any significant data differences between the groups of samples. This was followed by
a principal component analysis (PCA) for an easier and more thorough understanding
of any possible relationships between the studied samples regarding their phenolic and
volatile compounds contents. Further, a cluster analysis (CA) was carried out to detect any
Foods 2022,11, 303 6 of 23
similarities between the samples. The statistical significance was set at p< 0.05 and the
results were processed using the software Statistica 12.5 (StatSoft, Inc., Tulsa, OK, USA).
3. Results and Discussion
3.1. Vinegar Production
The juice employed for the production of prickly pear vinegar, was first characterized.
The initial sugar content was equivalent to 14.24
◦
Brix or 7.92
◦
Baumé, with 6.06 pH,
a density of 1.055 g/mL and a total acidity of 0.82 g/100 mL expressed as citric acid. When
the alcoholic fermentation was accomplished, the alcohol content was 5.27% (v/v) and the
◦
Brix value was around 1
◦
. At that moment, the sugar content was increased until 14
◦
Brix
degrees was reached (the same as the juice initial
◦
Brix value) in order to obtain a greater
final alcohol content. The final alcohol content of prickly pear wine was 8.7% (v/v).
The acetic acid bacteria (AAB) strains that had presented thermo-ethanol tolerance
characteristics in a previous study [
32
] were selected to be used as the starter culture for the
acetic fermentation of the prickly pear wine at two different temperatures: 30
◦
C and 37
◦
C.
The wine at 30
◦
C was fermented for 2 months, whereas the one at 37
◦
C was fermented for
three months. The prickly pear vinegar produced by the surface culture at 30
◦
C reached a
higher acidity value compared to that reached by the vinegar produced at 37
◦
C. This was
probably due to the fact that at 30
◦
C all the bacteria from the Acetobacter genus had the
capacity to produce acetic acid, which resulted in a mean value of 5.87 g/100 mL. These
bacteria produced vinegars with a lower acidity compared to that of the vinegars that
had been elaborated using bacteria from the Gluconobacter genus or with the mixture of
bacteria obtained from Sherry vinegar (7.46 g/100 mL and 7.56 g/100 mL, respectively).
Contrariwise, at 37
◦
C none of these bacteria strains or combinations of strains produced
high concentrations of acetic acid, so that the acetobacter strains, the gluconobacter strains and
the mixed strains obtained from Sherry vinegar achieved similar concentrations of just 1.88,
1.90 and 1.89 g/100 mL, respectively. Several reasons could explain this lower productivity,
such as the effect of the high temperature on the viability of the acetic acid bacteria even if
the two genera displayed thermotolerant characteristics in a previous study [
32
]. Other
reasons could be the greater evaporation of ethanol during the fermentation at the highest
temperature or a poor tolerance to a large ethanol concentration in the wine (8.7%) when
at a high temperature. These are considered to be stressful conditions for acetic bacteria
growth [
15
]. Saeki et al. demonstrated that bacterial growth at 37
◦
C seemed to be almost
impossible and that ethanol consumption was largely delayed. In fact, the acetic bacteria
did not grow at 39
◦
C in a medium containing over 3% ethanol [
38
]. Perumpuli et al.
indicated that in the static fermentation processes, the lower oxygen availability in the
liquid culture could negatively affect the production of acetic acids [39].
3.2. Volatile Compounds
In the present study, a total of 85 individual volatile compounds from different families
have been determined and identified in different prickly pear samples (juice, wine and
vinegars) by Stir Bar Sorptive Extraction coupled to Gas Chromatography–Mass Spectrom-
etry (SBSE/GC-MS). In order to determine any statistical differences between the prickly
pear samples regarding their composition of volatile compounds, the data were submitted
to analysis of variance (ANOVA, p< 0.05).
Table 1indicates the mean relative areas of all the studied compounds in the three
studied matrices as well as the results from the ANOVA. ANOVA indicates if the variable
under study (matrix) has a significant effect on the concentration of the studied compounds,
but this statistical analysis does not indicate neither if there are differences in the concentra-
tions of the compounds for all the levels of the studied variable, nor which level presents
the higher or lower value. To obtain that information, a post hoc test such as the Tukey test
was employed. This test compares all possible pairs of means and indicates which ones
are different compared with each other. These results are also presented in the table by
showing different letters in the same row for each analyzed compound.
Foods 2022,11, 303 7 of 23
Table 1.
Retention times (RT), mean relative areas and standard deviations (SD) of volatile compounds
identified by SBSE-GC-MS in different prickly pear matrices (juice, wine, vinegar). Results of analysis
of variance taking into account the matrix.
Compounds RT Juice Wine Vinegar ANOVA
Mean ±SD Mean ±SD Mean ±SD F Ratio p-Value
Ethyl acetate 8.89 0.3421 ±0.0428 a 13.96 ±1.169 b 0.1669 ±0.2122 a 3126 0.0000 *
1,3-Dioxolane, 2,4,5-trimethyl- 11.72 ND a 0.1049 ±0.0517 b 0.0089 ±0.0256 a 13.5 0.0000 *
Diacetyl 13.08 0.0055 ±0.0003 ND 0.0093 ±0.0130 0.590 0.5561
Isobutyl acetate 15.00 0.0058 ±0.0002 a 0.0915 ±0.0004 b 0.0047 ±0.0105 a 68.4 0.0000 *
Hexanal 18.01 0.0110 ±0.0001 b 0.0028 ±0.0002 a 0.0026 ±0.0011 a 56.7 0.0000 *
2-methyl-1-propanol 19.04 0.0022 ±0.0004 a 0.0334 ±0.0003 b 0.0027 ±0.0062 a 24.2 0.0000 *
Isoamyl acetate 19.74 0.0555 ±0.0161 a 0.8122 ±0.0257 b 0.0402 ±0.0756 a 104 0.0000 *
Acetic acid, pentyl ester 21.59 0.0321 ±0.0017 c 0.0093 ±0.0003 b ND a 30,820 0.0000 *
2,6-dimethyl-4-heptanone 21.69 0.0062 ±0.0005 0.0086 ±0.0011 0.0045 ±0.0047 0.863 0.4254
2-methyl-1-butanol 23.11 0.0032 ±0.0025 a 0.4001 ±0.0296 b 0.0234 ±0.0442 a 72.6 0.0000 *
3-methyl-1-butanol 23.24 0.0050 ±0.0005 a 0.3833 ±0.0314 b 0.0279 ±0.0473 a 56.6 0.0000 *
Furan, 2-pentyl- 23.67 0.0068 ±0.0009 b ND a ND a 4884 0.0000 *
Hexanoic acid, ethyl ester 23.85 0.0115 ±0.0017 a 0.0287 ±0.0257 b 0.0035 ±0.0042 a 27.6 0.0000 *
Styrene 24.50 0.0015 ±0.0002 b 0.0075 ±0.0031 c 0.0003 ±0.0002 a 331 0.0000 *
1-pentanol 24.58 0.0039 ±0.0001 c 0.0025 ±0.0002 b 0.0001 ±0.0003 a 220 0.0000 *
Hexyl acetate 25.50 0.0460 ±0.0055 c 0.0108 ±0.0027 b 0.0012 ±0.0028 a 255 0.0000 *
Acetoin 25.72 ND ND 0.1369 ±0.1829 1.09 0.3384
Acetol 26.08 0.0079 ±0.0008 a 0.0489 ±0.0150 b 0.0085 ±0.0068 a 33.2 0.0000 *
2-octanone 26.12 0.0068 ±0.0005 b 0.0132 ±0.0031 c ND a 1904 0.0000 *
3-Hexen-1-ol, acetate, (Z) 26.90 0.0054 ±0.0000 b ND a ND a 1.30 ×
1090.0000 *
E-3-hexenyl acetate 26.91 0.0054 ±0.0001 c 0.0028 ±0.0004 b ND a 15,409 0.0000 *
2-Hexen-1-ol, acetate, (E) 27.45 0.0304 ±0.0032 b ND a ND a 7647 0.0000 *
Ethyl lactate 27.55 0.0005 ±0.0000 a 0.2038 ±0.0148 ab 0.3701 ±0.1801 b 4.98 0.0089 *
1-hexanol 28.14 0.0288 ±0.0009 b 0.0334 ±0.0028 b 0.0006 ±0.0019 a 473 0.0000 *
3-Hexen-1-ol, (E)- 28.41 0.0033 ±0.0005 b 0.0046 ±0.0021 c 0.0001 ±0.0005 a 96.4 0.0000 *
3-Hexen-1-ol, (Z)- 29.20 0.0025 ±0.0003 b 0.0022 ±0.0001 b 0.0002 ±0.0005 a 34.4 0.0000 *
2-Hexen-1-ol, (E)- 29.89 0.0072 ±0.0007 b ND a ND a 8389 0.0000 *
Acetic acid 30.79 0.0444 ±0.0003 0.0503 ±0.0043 0.2439 ±0.2641 1.08 0.3428
2-octenal 31.31 0.0268 ±0.0040 b ND a ND a 3772 0.0000 *
Octanoic acid, ethyl ester 31.46 ND a 0.1203 ±0.0103 b 0.0007 ±0.0031 a 1306 0.0000 *
trans-linalooloxide 31.60 ND ND 0.0079 ±0.0054 4.20 0.0180 *
1-heptanol 31.82 0.0033 ±0.0001 b 0.0056 ±0.0008 c ND a 5231 0.0000 *
2,4-heptadienal, (E,E)- 32.29 0.0037 ±0.0010 b ND a ND a 1129 0.0000 *
cis-linalooloxide 32.61 0.0003 ±0.0000 a 0.0013 ±0.0005 b 0.0056 ±0.0015 c 20.5 0.0000 *
4-Octenoic acid, ethyl ester, (Z)- 32.73 ND a 0.0041 ±0.0001 b ND a 189,066 0.0000 *
1-Hexanol, 2-ethyl- 33.05 0.0016 ±0.0001 0.0097 ±0.0009 0.0160 ±0.0139 1.25 0.2914
Cyclopentene 34.17 0.0012 ±0.0001 b 0.0011 ±0.0001 b 0.0001 ±0.0002 a 34.2 0.0000 *
Benzaldehyde 34.41 0.0012 ±0.0001 a 0.0015 ±0.0001 a 0.0118 ±0.0064 b 5.17 0.0075 *
2,3-butanediol 34.67 0.0010 ±0.0002 a 0.0059 ±0.0008 ab 0.0151 ±0.0069 b 5.73 0.0046 *
Linalool 35.02 0.0190 ±0.0007 b 0.0673 ±0.0007 c 0.0012 ±0.0017 a 1671 0.0000 *
Nonenal 35.14 0.0075 ±0.0008 b ND a ND a 6850 0.0000 *
Isobutyric acid 35.40 0.0047 ±0.0006 0.0221 ±0.0012 0.0164 ±0.0157 0.705 0.4969
1-octanol 35.53 0.0079 ±0.0006 b 0.0339 ±0.0005 c 0.0010 ±0.0022 a 222 0.0000 *
trans-2-cis-6-nonadienal 36.82 0.0117 ±0.0008 b 0.0314 ±0.0023 c 0.0001 ±0.0004 a 4988 0.0000 *
trans-2-Decenol 37.43 0.0066 ±0.0001 b ND a ND a
1,139,269
0.0000 *
Butanoic acid 37.65 ND ND 0.0011 ±0.0014 1.17 0.3135
Sulfide, allyl methyl 38.12 0.0022 ±0.0001 a 0.0115 ±0.0005 c 0.0043 ±0.0027 b 8.08 0.0006 *
Decanoic acid, ethyl ester 38.82 ND a 0.0248 ±0.0022 b ND a 10,625 0.0000 *
Isovaleric acid 39.17 ND ND 0.0341 ±0.0390 1.50 0.2288
1-nonanol 39.23 0.0180 ±0.0004 b 0.1010 ±0.0064 c 0.0039 ±0.0041 a 557 0.0000 *
Butanedioic acid, diethyl ester 39.62 ND a 0.0166 ±0.0007 ab 0.0259 ±0.0099 b 7.60 0.0009 *
trans, cis-2,6-nonadienyl acetate 40.23 0.0220 ±0.0030 b ND a ND a 4479 0.0000 *
α-Terpineol 40.76 0.0048 ±0.0001 a 0.0235 ±0.0005 b 0.0083 ±0.0044 a 12.7 0.0000 *
2-Nonen-1-ol, (E)- 41.13 0.0518 ±0.0029 c ND a 0.0017 ±0.0044 b 128 0.0000 *
cis-6-nonenol 41.22 0.0394 ±0.0026 a 0.2145 ±0.0073 b 0.0221 ±0.0291 a 44.0 0.0000 *
Benzyl acetate 41.59 0.0131 ±0.0042 0.0046 ±0.0002 0.0057 ±0.0054 1.91 0.1536
β-Citronellol 42.96 0.1333 ±0.0019 c 0.0239 ±0.0014 b 0.0028 ±0.0014 a 8759 0.0000 *
trans, cis-2,6-Nonadien-1-ol 42.96 0.1340 ±0.0011 b ND a ND a
1,226,155
0.0000 *
Methyl salicylate 43.47 0.0196 ±0.0012 b 0.0256 ±0.0002 c 0.0011 ±0.0011 a 711 0.0000 *
Ethyl phenylacetate 43.60 0.0027 ±0.0000 0.0223 ±0.0003 0.0145 ±0.0189 0.568 0.5687
Phenethyl acetate 44.78 0.0383 ±0.0049 0.1995 ±0.0011 0.2040 ±0.2131 0.605 0.5481
2,4-decadienal 44.98 0.0065 ±0.0019 b ND a ND a 1056 0.0000 *
Foods 2022,11, 303 8 of 23
Table 1. Cont.
Compounds RT Juice Wine Vinegar ANOVA
Mean ±SD Mean ±SD Mean ±SD F Ratio p-Value
β-damascenone 45.24 ND a 0.0075 ±0.0048 b ND a 206 0.0000 *
Hexanoic acid 45.41 0.0048 ±0.0002 0.0119 ±0.0012 0.0183 ±0.0126 1.37 0.2572
Geraniol 45.74 0.0067 ±0.0004 b 0.0167 ±0.0001 c 0.0013 ±0.0021 a 58.0 0.0000 *
cis-geranylacetone 46.28 0.0018 ±0.0002 a 0.0129 ±0.0012 b 0.0026 ±0.0037 a 7.63 0.0009 *
Benzyl alcohol 46.58 0.0043 ±0.0001 a 0.0072 ±0.0001 a 0.0110 ±0.0023 b 11.3 0.0000 *
Benzenepropanoic acid, ethyl ester
47.18 ND a 0.0420 ±0.0018 b 0.0054 ±0.0062 a 36.0 0.0000 *
Phenylethyl alcohol 47.88 0.0096 ±0.0011 a 0.1723 ±0.0041 ab 0.2644 ±0.1385 b 3.77 0.0267 *
3-Phenyl-1-propanol, acetate 49.09 0.0038 ±0.0015 0.0092 ±0.0008 0.0082 ±0.0155 0.084 0.9194
2,4-Decadien-1-ol 50.65 0.0030 ±0.0004 b ND a ND a 5293 0.0000 *
Phenol 50.73 0.0012 ±0.0004 a 0.0045 ±0.0008 b 0.0035 ±0.0008 b 8.41 0.0005 *
4-hydroxynonanoic acid lactone 52.19 0.0225 ±0.0014 a 0.0315 ±0.0003 a 0.0521 ±0.0073 b 23.6 0.0000 *
Benzenepropanol 52.31 ND a 0.0046 ±0.0004 ab 0.0050 ±0.0023 b 4.63 0.0122 *
Octanoic acid 52.57 0.0275 ±0.0023 a 0.1683 ±0.0031 b 0.1106 ±0.0525 ab 3.78 0.0264 *
Ethyl cinnamate 55.18 ND a 0.0135 ±0.0000 b 0.0031 ±0.0019 a 34.1 0.0000 *
Cinnamyl acetate 55.67 0.0030 ±0.0007 b 0.0010 ±0.0001 a 0.0004 ±0.0003 a 58.3 0.0000 *
Nonanoic acid 55.92 0.0136 ±0.0003 a 0.0817 ±0.0008 b 0.0432 ±0.0269 ab 3.34 0.0398 *
Thymol 56.40 0.0027 ±0.0001 a 0.0146 ±0.0003 b 0.0045 ±0.0013 a 59.4 0.0000 *
2-Octenoic acid 56.43 0.0042 ±0.0004 b ND a ND a 10,244 0.0000 *
Decanoic acid 59.15 0.0253 ±0.0118 a 0.1164 ±0.0007 b 0.0256 ±0.0176 a 26.4 0.0000 *
2-nonenoic acid 59.62 0.0089 ±0.0007 b 0.0016 ±0.0004 a 0.0024 ±0.0016 a 17.2 0.0000 *
Dihydromethyl jasmonate 59.95 ND a ND a 0.0013 ±0.0010 b 3.35 0.0396 *
G-dodecalactone 63.08 0.0136 ±0.0031 a 0.0530 ±0.0008 b 0.0169 ±0.0055 a 43.4 0.0000 *
Dodecanoic acid 66.58 0.0166 ±0.0086 a 0.1231 ±0.0053 b 0.0082 ±0.0089 a 165 0.0000 *
Tetradecanoic acid 78.46 0.0052 ±0.0004 a 0.0123 ±0.0014 b 0.0024 ±0.0025 a 17.0 0.0000 *
Different letters in the same row indicate significant differences according to Tukey’s test (p< 0.05). * Values are
significant at p< 0.05; ND: Not detected.
Some of the volatile compounds identified in this research had been previously iden-
tified in other fruit vinegars such as lemon vinegar [
33
], orange vinegar [
40
], tomato
vinegar [41], strawberry vinegar or persimmon vinegar [42], among others.
From a general point of view, almost all the volatile compounds showed significant
differences according to the matrix (p< 0.05). This was an expected result because during
the vinegar production, two different fermentation processes take place (alcoholic and
acetic fermentation), and many compounds are produced and modified other than ethanol
and acetic acid [
15
]. Some of the compounds, such as certain alcohols and esters such as
2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-1-propanol, isobutyl acetate or hexanoic
acid ethyl ester, among others, increased significantly during the alcoholic fermentation
and when the acetic fermentation was accomplished, they decreased. These results are
in accordance with those reported by previous research works [
33
,
40
,
41
]. On the other
hand, other compounds such as nonenal, 2-octanone, acetic acid pentyl ester, E-3-hexenyl
acetate, 2 octenal and 2,4-decadienal among others, disappeared during the transformation
process from prickly pear juice into prickly pear vinegar. It should also be noted that many
compounds are normally degraded during the acetic fermentation process that produces
vinegar [
15
]. It was found that some volatile compounds such as hexanal, 1-pentanol,
hexyl acetate or
β
-citronellol were the majority compounds in prickly pear juice, which
would explain them being detected in the prickly pear vinegar, although at a lower content.
Their presence in the prickly pear vinegar could also be due to the lesser degradation
of compounds that takes place during acetic fermentation in surface culture processes
when compared to the submerged culture method [
15
]. On the other hand, some of the
compounds that were not detected in prickly pear juice were formed during the acetic
fermentation. Some of them were trans-linalol oxide, dihydromethyl jasmonate, benzene-
propanol, butanedioic acid diethyl ester, octanoic acid ethyl ester and benzenepropanoic
acid ethyl ester, among others. Other authors who studied the production of strawberry or
persimmon vinegar also found an increase in certain volatile compounds resulting from the
influence of the microorganisms employed in the fermentation process [
42
]. Furthermore,
the production of certain volatile compounds could be favored by the employment of
Foods 2022,11, 303 9 of 23
inoculated yeasts for the alcoholic fermentation, instead of native yeasts [
43
]. The results
obtained in this study on the volatile composition of prickly pear juice were in agreement
with those reported by Arena et al. [
20
]. These researchers found that the major class of
volatile compounds in prickly pear fruit were the alcohols represented by trans-2-hexen-1-ol
and n-hexanol, even though numerous esters and carbonyl compounds were also present
at low concentrations. No data on prickly pear wine or vinegar regarding their volatile
composition have been found in the literature.
In order to corroborate the differences that took place during the vinegar production
process, the data obtained were submitted to a multivariate statistical study (principal
component analysis—PCA). This analysis allowed us to identify eight PCs that could
explain 94.61% of the total variability (eigenvalues > 1). Figure 1represents the distribution
of the samples on the plane defined by the first two PCs.
Foods 2022, 11, x FOR PEER REVIEW 10 of 25
Figure 1. PCA of volatile compounds. Distribution of all samples onto the plane defined by the first
two PCs. (A): original graph; (B): zoomed area. J: prickly pear juice; W: prickly pear wine; V: prickly
pear vinegar.
As can be seen, PC1 allowed to distinguish vinegar samples (V) from juice (J) and
wine (W), whereas PC2 was able to separate the three prickly pear matrixes. The volatile
compounds that contributed more and with a greater influence on PC1 were as follows:
methyl salicylate, 1-hexanol, 1-heptanol, 2-octanone, 1-pentanol, trans-2-cis-6-nonadienal
and 3-hexen-1-ol (E). Regarding to PC2, these ones were: 3-hexen-1-ol acetate (Z), trans-2-
decenol, trans, cis-2,6-nonadien-1-ol, 2-octenoic acid, 2-hexen-1-ol acetate (E), 2-hexen-1-
ol (E) and nonenal.
To study the effect from certain parameters, such as fermentation temperature and
acetic acid bacteria on the volatile composition of the prickly pear vinegar, the prickly
Figure 1.
PCA of volatile compounds. Distribution of all samples onto the plane defined by the first two
PCs. (
A
): original graph; (
B
): zoomed area. J: prickly pear juice; W: prickly pear wine; V: prickly pear vinegar.
Foods 2022,11, 303 10 of 23
As can be seen, PC1 allowed to distinguish vinegar samples (V) from juice (J) and
wine (W), whereas PC2 was able to separate the three prickly pear matrixes. The volatile
compounds that contributed more and with a greater influence on PC1 were as follows:
methyl salicylate, 1-hexanol, 1-heptanol, 2-octanone, 1-pentanol, trans-2-cis-6-nonadienal
and 3-hexen-1-ol (E). Regarding to PC2, these ones were: 3-hexen-1-ol acetate (Z), trans-2-
decenol, trans, cis-2,6-nonadien-1-ol, 2-octenoic acid, 2-hexen-1-ol acetate (E), 2-hexen-1-ol
(E) and nonenal.
To study the effect from certain parameters, such as fermentation temperature and
acetic acid bacteria on the volatile composition of the prickly pear vinegar, the prickly pear
wine was inoculated with a different genus of acetic acid bacteria (Acetobacter,Gluconobacter
and a mixture of bacteria from Sherry vinegar) and incubated at 30
◦
C and 37
◦
C. Table 2
shows the results from the analysis of variance considering temperature and genus as the
independent variables and volatile compounds as the dependent ones. As can be seen, most
of the volatile compounds were significantly affected by fermentation temperature, whereas
no significant differences between the volatile composition of the vinegars produced by
the different bacteria inoculum were detected. Hence, the type of acetic acid bacteria
inoculated does not seem to be relevant with respect to the volatile composition of the
vinegars. Other studies have reported slight differences in the taste of vinegars produced
using Gluconobacter genus bacteria when compared to those produced using Acetobacter,
since the former ones generate greater amounts of gluconate [
44
]. Kim et al. observed that
the production of certain metabolites in tomato vinegar depended on the strain of acetic
acid bacteria used and that the fermentation temperature also had a significant influence
on the production of such metabolites [45].
Table 2.
Analysis of variance considering the effect of temperature (30
◦
C/37
◦
C) and bacteria
inoculum (Acetobacter (A)/Gluconobacter (G)/Mixture of bacteria (M)) on volatile compounds of
prickly pear vinegar produced by surface culture.
Compounds Temperature (30 ◦C/37 ◦C) Bacteria Inoculum (A/G/M)
F Ratio p-Value F Ratio p-Value
Ethyl acetate 0.076 0.7833 0.425 0.6546
1,3-Dioxolane, 2,4,5-trimethyl 11.4 0.0011 * 0.980 0.3795
Diacetyl 35.3 0.0000 * 0.582 0.5610
Isobutyl acetate 0.057 0.8108 0.196 0.8219
Hexanal 11.4 0.0011 * 2.24 0.1126
2-methyl-1-propanol 19.1 0.0000 * 0.070 0.9317
Isoamyl acetate 0.016 0.8987 0.374 0.6885
2,6-dimethyl-4-heptanone 6.91 0.0102 * 0.281 0.7551
2-methyl-1-butanol 17.0 0.0001 * 1.39 0.2524
3-methyl-1-butanol 24.4 0.0000 * 0.486 0.6167
Hexanoic acid, ethyl ester 0.766 0.3837 0.801 0.4522
Styrene 1.10 0.2962 0.967 0.3844
1-pentanol 10.0 0.0021 * 0.926 0.4000
Hexyl acetate 13.0 0.0005 * 7.46 0.0010*
Acetoin 59.1 0.0000 * 1.16 0.3161
Acetol 0.308 0.5799 2.44 0.0927
Ethyl lactate 0.740 0.3921 2.92 0.0593
1-hexanol 8.47 0.0046 * 0.412 0.6635
3-Hexen-1-ol, (E)- 5.08 0.0267 * 0.945 0.3927
3-Hexen-1-ol, (Z)- 12.8 0.0006 * 0.622 0.5392
Acetic acid 47.6 0.0000 * 1.43 0.2443
Octanoic acid, ethyl ester 0.008 0.9286 0.833 0.4382
trans-linalooloxide 353 0.0000 * 0.091 0.9127
cis-linalooloxide 234 0.0000 * 0.100 0.9042
1-Hexanol, 2-ethyl- 4378 0.0000 * 0.045 0.9558
Cyclopentene 11.0 0.0013 * 0.578 0.5628
Benzaldehyde 24.6 0.0000 * 4.33 0.0161 *
Foods 2022,11, 303 11 of 23
Table 2. Cont.
Compounds Temperature (30 ◦C/37 ◦C) Bacteria Inoculum (A/G/M)
F Ratio p-Value F Ratio p-Value
2,3-butanediol 23.2 0.0000 * 1.40 0.2504
Linalool 45.4 0.0000 * 0.639 0.5303
Isobutyric acid 60.3 0.0000 * 1.30 0.2777
1-octanol 16.3 0.0001 * 0.907 0.4075
trans-2-cis-6-nonadienal 12.3 0.0007 * 1.07 0.3465
Butanoic acid 74.5 0.0000 * 0.959 0.3871
Sulfide, allyl methyl 3.75 0.0559 1.64 0.1992
Isovaleric acid 50.9 0.0000 * 2.05 0.1350
1-nonanol 10.7 0.0015 * 2.10 0.1279
Butanedioic acid, diethyl ester 39.1 0.0000 * 1.44 0.2407
α-terpineol 490 0.0000 * 0.490 0.6140
2-Nonen-1-ol, (E)- 4.63 0.0342 * 3.05 0.0523
cis-6-nonenol 6.50 0.0126 * 1.02 0.3623
Benzyl acetate 31.0 0.0000 * 2.66 0.0755
β-Citronellol 30.3 0.0000 * 2.34 0.1026
Methyl salicylate 72.0 0.0000 * 2.77 0.0682
Ethyl phenylacetate 15.8 0.0001 * 0.847 0.4322
Phenethyl acetate 34.4 0.0000 * 0.050 0.9509
Hexanoic acid 35.6 0.0000 * 2.35 0.1007
Geraniol 29.8 0.0000 * 1.95 0.1480
cis-geranylacetone 0.008 0.9261 0.229 0.7954
Benzyl alcohol 0.875 0.3522 3.33 0.0404 *
Benzenepropanoic acid, ethyl ester 66.9 0.0000 * 0.394 0.6754
Phenylethyl alcohol 1.14 0.2874 2.18 0.1191
3-Phenyl-1-propanol, acetate 11.1 0.0013 * 0.767 0.4676
Phenol 21.0 0.0000 * 0.559 0.5735
4-hydroxynonanoic acid lactone 20.6 0.0000 * 0.318 0.7286
Benzenepropanol 35.0 0.0000 * 1.94 0.1489
Octanoic acid 15.4 0.0002 * 2.86 0.0624
Ethyl cinnamate 257 0.0000 * 1.20 0.3047
Cinnamyl acetate 42.1 0.0000 * 0.429 0.6521
Nonanoic acid 33.1 0.0000 * 2.07 0.1326
Thymol 41.2 0.0000 * 1.35 0.2638
Decanoic acid 42.6 0.0000 * 1.52 0.2239
2-nonenoic acid 31.0 0.0000 * 3.47 0.0354 *
Dihydromethyl jasmonate 106 0.0000 * 0.530 0.5904
G-Dodecalactone 72.4 0.0000 * 1.17 0.3139
Dodecanoic acid 56.3 0.0000 * 1.10 0.3348
Tetradecanoic acid 75.8 0.0000 * 0.436 0.6477
* Values are significant at p< 0.05.
Table 3indicates the mean relative areas of the volatile compounds in the vinegars pro-
duced at 30
◦
C and at 37
◦
C. It can be observed that most of the volatile compounds reached
higher concentration values in the vinegar that had been produced at 30
◦
C compared to
those corresponding to the vinegar produced at 37
◦
C. According to Liu et al., the aroma
compounds and their concentrations were influenced by the fermentation temperature, so
that as the temperature was increased, the number of volatiles would decrease [
46
]. Conse-
quently, certain compounds, such as 1,3-dioxolane, 2,4,5-trimethyl-, 1-hexanol, 3-hexen-1-ol
(E), 3-hexen-1-ol (Z), cyclopentene or trans-2-cis-6-nonadienal, would even disappear when
the acetic fermentation was conducted at 37
◦
C. These changes in the concentration of
certain compounds could be explained by their possible reduction by evaporation when
submitted to high temperatures (37
◦
C), which, in some cases, might lead to their total
disappearance [
15
]. For example, cyclopentene and 1,3-dioxolane have boiling points at
atmospheric pressure of 44
◦
C and 74
◦
C, respectively; however, the mean relative area
detected for some compounds, such as trans-linalooloxide, cis-linalooloxide, 1-hexanol
Foods 2022,11, 303 12 of 23
2-ethyl-, benzaldehyde, 1-nonanol and dihydromethyl jasmonate among others was greater
at 37
◦
C than at 30
◦
C. This was probably due to changes in the metabolism of the bacteria
as the temperature varied. Thus, the production of 2,3-butanediol and butanoic acid during
the elaboration of tomato vinegar at different temperatures was studied by Kim et al. [
45
]
and 2,3-butanediol was detected at greater concentrations in those vinegars produced
at a higher temperature (34
◦
C), whereas butanoic acid was only found in the vinegars
produced at 30
◦
C. In the present study, similar behavior by both of these compounds
was observed. Although the increment of the fermentation temperature led, as expected,
to a general reduction in the volatile content in the vinegars, partial fermentations were
accomplished at 37
◦
C even if the optimal growth temperature for acetic acid bacteria is
between 25 and 30 ◦C [15].
Table 3.
Mean relative areas and standard deviations of volatile identified by SBSE-GC-MS in Prickly
pear Vinegar produced by surface culture method at 30 ◦C and 37 ◦C.
Compounds 30 ◦C 37 ◦C
Mean ±SD Mean ±SD
Ethyl acetate 0.1607 ±0.1160 0.1733 ±0.2798
1,3-dioxolane, 2,4,5-trimethyl- 0.0176 ±0.0340 b ND a
Diacetyl 0.0162 ±0.0149 b 0.0022 ±0.0042 a
Isobutyl acetate 0.0045 ±0.0028 0.0050 ±0.0147
Hexanal 0.0030 ±0.0010 b 0.0023 ±0.0011 a
2-methyl-1-propanol 0.0053 ±0.0080 b 0.0000 ±0.0001 a
Isoamyl acetate 0.0391 ±0.0317 0.0412 ±0.1034
2,6-dimethyl-4-heptanone 0.0058 ±0.0055 b 0.0032 ±0.0034 a
2-methyl-1-butanol 0.0411 ±0.0568 b 0.0052 ±0.0062 a
3-meth-1-butanol 0.0499 ±0.0586 b 0.0054 ±0.0066 a
Hexanoic acid, ethyl ester 0.0031 ±0.0033 0.0039 ±0.0049
Styrene 0.0003 ±0.0003 0.0002 ±0.0002
1-pentanol 0.0002 ±0.0004 b 0.0000 ±0.0001 a
Hexyl acetate 0.0022 ±0.0036 b 0.0002 ±0.0004 a
Acetoin 0.2520 ±0.1984 b 0.0191 ±0.0101 a
Acetol 0.0089 ±0.0044 0.0081 ±0.0087
Ethyl lactate 0.3537 ±0.2461 0.3869 ±0.0617
1-hexanol 0.0012 ±0.0026 b ND a
3-Hexen-1-ol, (E)- 0.0002 ±0.0007 b ND a
3-Hexen-1-ol, (Z)- 0.0004 ±0.0007 b ND a
Acetic acid 0.3995 ±0.2952 b 0.0847 ±0.0484 a
Octanoic acid, ethyl ester 0.0007 ±0.0029 0.0007 ±0.0034
trans-linalooloxide 0.0031 ±0.0031 a 0.0127 ±0.0013 b
cis-linalooloxide 0.0043 ±0.0007 a 0.0068 ±0.0009 b
1-Hexanol, 2-ethyl- 0.0025 ±0.0010 a 0.0298 ±0.0025 b
Cyclopentene 0.0002 ±0.0003 b ND a
Benzaldehyde 0.0088 ±0.0073 a 0.0149 ±0.0033 b
2,3-butanediol 0.0120 ±0.0051 a 0.0184 ±0.0071 b
Linalool 0.0022 ±0.0018 b 0.0003 ±0.0005 a
Isobutyric acid 0.0263 ±0.0168 b 0.0063 ±0.0020 a
1-octanol 0.0019 ±0.0029 b 0.0001 ±0.0005 a
Trans-2-cis-6-nonadienal 0.0003 ±0.0005 b ND a
Butanoic acid 0.0020 ±0.0014 b 0.0001 ±0.0003 a
Sulfide, allyl methyl 0.0049 ±0.0034 0.0038 ±0.0013
Isovaleric acid 0.0576 ±0.0433 b 0.0101 ±0.0048 a
1-nonanol 0.0025 ±0.0051 a 0.0053 ±0.0021 b
Butanedioic acid, diethyl ester 0.0204 ±0.0112 a 0.0314 ±0.0029 b
α-terpineol 0.0123 ±0.0023 b 0.0042 ±0.0007 a
2-Nonen-1-ol, (E)- 0.0027 ±0.0060 b 0.0007 ±0.0012 a
cis-6-nonenol 0.0297 ±0.0395 b 0.0143 ±0.0032 a
Benzyl acetate 0.0085 ±0.0065 b 0.0029 ±0.0003 a
β-Citronellol 0.0035 ±0.0015 b 0.0021 ±0.0007 a
Foods 2022,11, 303 13 of 23
Table 3. Cont.
Compounds 30 ◦C 37 ◦C
Mean ±SD Mean ±SD
Methyl salicylate 0.0019 ±0.0012 b 0.0004 ±0.0001 a
Ethyl phenylacetate 0.0218 ±0.0245 b 0.0069 ±0.0015 a
Phenethyl acetate 0.3165 ±0.2540 b 0.0890 ±0.0103 a
Hexanoic acid 0.0250 ±0.0149 b 0.0114 ±0.0018 a
Geraniol 0.0024 ±0.0026 b 0.0002 ±0.0003 a
cis-geranylacetone 0.0027 ±0.0017 0.0026 ±0.0051
Benzyl alcohol 0.0108 ±0.0031 0.0112 ±0.0009
Benzenepropanoic acid, ethyl ester 0.0094 ±0.0064 b 0.0012 ±0.0012 a
Phenylethyl alcohol 0.2801 ±0.1939 0.2484 ±0.0158
3-Phenyl-1-propanol, acetate 0.0134 ±0.0207 b 0.0029 ±0.0006 a
Phenol 0.0038 ±0.0010 b 0.0031 ±0.0005 a
4-hydroxynonanoic acid lactone 0.0489 ±0.0078 a 0.0554 ±0.0051 b
Benzenepropanol 0.0037 ±0.0027 a 0.0062 ±0.0006 b
Octanoic acid 0.1309 ±0.0676 b 0.0899 ±0.0099 a
Ethyl cinnamate 0.0047 ±0.0013 b 0.0015 ±0.0004 a
Cinnamyl acetate 0.0003 ±0.0004 a 0.0006 ±0.0001 b
Nonanoic acid 0.0572 ±0.0321 b 0.0289 ±0.0033 a
Thymol 0.0052 ±0.0014 b 0.0037 ±0.0006 a
Decanoic acid 0.0356 ±0.0198 b 0.0153 ±0.0046 a
2-nonenoic acid 0.0032 ±0.0019 b 0.0015 ±0.0005 a
Dihydromethyl jasmonate 0.0006 ±0.0005 a 0.0020 ±0.0008 b
G-dodecalactone 0.0206 ±0.0053 b 0.0131 ±0.0022 a
Dodecanoic acid 0.0137 ±0.0085 b 0.0025 ±0.0048 a
Tetradecanoic acid 0.0041 ±0.0023 b 0.0007 ±0.0011 a
Different letters in the same row indicate significant differences according to Tukey’s test (p< 0.05); ND: Not detected.
The resulting data were submitted to a multivariate statistical study (principal compo-
nent analysis, PCA), which allowed us to identify 11 PCs that explained 92.7% of the total
variability (eigenvalues > 1). Figure 2shows the distribution of all the vinegar samples on
the plane defined by PC1 and PC2. As it can be seen, PC1 separated the vinegars produced
at 37
◦
C from some of the vinegars fermented at 30
◦
C, which were mainly those vinegars
that had been fermented using gluconobacter. PC2, on the other hand, was able to separate
all the vinegars fermented at 37
◦
C from those fermented at 30
◦
C. PC1 was mainly related
to the presence of acids, such as hexanoic acid, nonanoic acid, isovaleric acid, isobutyric
acid and acetic acid, while PC2 was related to certain alcohols, such as 1-octanol, linalool,
3-methyl-1-butanol, 3-hexen-1-ol (Z) or 1-pentanol.
To corroborate these results, the set of data was also submitted to a hierarchical
agglomerative cluster analysis (Figure 3). The squared Euclidean distance as metric and the
Ward method as the amalgamation rule were employed to set up the clusters. As illustrated,
three groups were obtained: vinegars produced by acetobacter at 30
◦
C and 37
◦
C (A 30 and
A 37), vinegars produced by the three types of bacteria inoculum at 37
◦
C (A/G/M 37) and
vinegars produced by the three types of bacteria inoculum at 30
◦
C (A/G/M 30). These
groupings revealed that the influence that the acetification temperature exerted on the
vinegars’ volatile profiles was quite high and even more significant than that corresponding
to the particular acetic acid bacteria inocula employed.
3.3. Phenolic Compounds and Antioxidant Activity
3.3.1. Phenolic Composition
In the present study, a total of 16 polyphenolic compounds in prickly pear samples
(juice, wine and vinegar) have been studied. To determine the statistical differences be-
tween prickly pear samples regarding their polyphenolic compounds content, the data
were submitted to analysis of variance (ANOVA, p< 0.05). Table 4displays the mean
concentrations of the different polyphenolic compounds depending on the matrix.
Foods 2022,11, 303 14 of 23
Foods 2022, 11, x FOR PEER REVIEW 15 of 25
Figure 2. PCA on volatile compounds. Distribution of all vinegar samples onto the plane defined by
the first two PCs. (A): original graph; (B): zoomed area. A 30/A 37: Acetobacter at 30 °C and 37 °C, G
30/G 37: Gluconobacter at 30 °C and 37 °C, M 30/M 37: Mixture of bacteria at 30 °C and 37 °C.
To corroborate these results, the set of data was also submitted to a hierarchical ag-
glomerative cluster analysis (Figure 3). The squared Euclidean distance as metric and the
Ward method as the amalgamation rule were employed to set up the clusters. As illus-
trated, three groups were obtained: vinegars produced by acetobacter at 30 °C and 37 °C
(A 30 and A 37), vinegars produced by the three types of bacteria inoculum at 37 °C
Figure 2.
PCA on volatile compounds. Distribution of all vinegar samples onto the plane defined by
the first two PCs. (
A
): original graph; (
B
): zoomed area. A 30/A 37: Acetobacter at 30
◦
C and 37
◦
C,
G 30/G 37: Gluconobacter at 30 ◦C and 37 ◦C, M 30/M 37: Mixture of bacteria at 30 ◦C and 37 ◦C.
Foods 2022,11, 303 15 of 23
Foods 2022, 11, x FOR PEER REVIEW 16 of 25
(A/G/M 37) and vinegars produced by the three types of bacteria inoculum at 30 °C
(A/G/M 30). These groupings revealed that the influence that the acetification temperature
exerted on the vinegars’ volatile profiles was quite high and even more significant than
that corresponding to the particular acetic acid bacteria inocula employed.
Figure 3. Cluster analysis taking into consideration the composition of volatile compounds of vine-
gar samples. A 30/A 37: Acetobacter at 30 °C and 37 °C, G 30/G 37: Gluconobacter at 30 °C and 37 °C,
M 30/M 37: Mixture of bacteria at 30 °C and 37 °C.
3.3. Phenolic Compounds and Antioxidant Activity
3.3.1. Phenolic Composition
In the present study, a total of 16 polyphenolic compounds in prickly pear samples
(juice, wine and vinegar) have been studied. To determine the statistical differences be-
tween prickly pear samples regarding their polyphenolic compounds content, the data
were submitted to analysis of variance (ANOVA, p < 0.05). Table 4 displays the mean con-
centrations of the different polyphenolic compounds depending on the matrix.
Ward`s method
Euclidean distanc es
A 30
A 30
A 30
A 30
A 37
A 37
A 37
A 30
G 30
A 30
A 30
A 30
A 37
A 37
A 37
A 37
A 37
A 37
A 37
A 37
A 37
A 37
A 37
M 37
M 37
A 37
A 37
G 37
G 37
G 37
G 37
M 30
A 30
A 30
A 30
A 30
A 30
G 30
A 30
A 30
G 30
M 30
A 30
G 30
0
2
4
6
8
10
Linkage Distance
Figure 3.
Cluster analysis taking into consideration the composition of volatile compounds of vinegar
samples. A 30/A 37: Acetobacter at 30
◦
C and 37
◦
C, G 30/G 37: Gluconobacter at 30
◦
C and 37
◦
C,
M 30/M 37: Mixture of bacteria at 30 ◦C and 37 ◦C.
Table 4.
Mean concentrations (ppm) and standard deviations of phenolic compounds identified by
UPLC-DAD in different prickly pear matrices (juice, wine, vinegar). Results of analysis of variance
taking into account the matrix.
Compounds Juice Wine Vinegar ANOVA
Mean ±SD Mean ±SD Mean ±SD F Ratio p-Value
Gallic acid 1.52 ±0.042 1.24 ±0.009 1.76 ±0.471 1.48 0.2334
Hydroxy-tyrosol 4.34 ±0.184 1.67 ±0.147 2.85 ±1.80 2.33 0.1035
Epigallocatechin ND a 5.93 ±0.172 b 8.26 ±1.45 b 34.3 0.0000 *
Tyrosol 57.1 ±2.54 42.4 ±1.74 56.2 ±9.62 2.10 0.1289
Vanillic acid ND ND 2.19 ±0.776 0.492 0.6125
Syringic acid 1.66 ±0.082 a 1.69 ±0.035 ab 2.32 ±0.376 b 5.65 0.0049 *
Ethyl gallate 3.57 ±0.233 c 1.91 ±0.164 b ND a 17373 0.0000 *
m-Coumaric acid 0.564 ±0.011 b ND a ND a 555,867 0.0000 *
Hesperidin 15.0 ±3.63 4.39 ±0.032 8.46 ±2.06 13.8 0.0000 *
Naringenin 4.56 ±0.879 c 2.47 ±0.011 a 3.68 ±0.908 b 2.73 0.0710
Protocatechualdehyde 0.956 ±0.030 a 1.28 ±0.027 ab 1.39 ±0.165 b 7.42 0.0010 *
Caffeic acid ND a ND a 1.06 ±0.100 b 220 0.0000 *
Ferulic acid 1.35 ±0.065 1.33 ±0.007 1.41 ±0.250 0.154 0.8581
Quercetin 1.30 ±0.441 1.13 ±0.117 1.33 ±0.309 0.452 0.6388
Cinnamic acid ND 0.048 ±0.020 0.125 ±0.073 3.70 0.0286 *
p-Hydroxybenzoic acid
9.86 ±0.469 b 1.33 ±0.160 a 1.14 ±0.450 a 370 0.0000 *
Different letters in the same row indicate significant differences according to Tukey’s test (p< 0.05). * Values are
significant at p< 0.05; ND: Not detected.
Some of the compounds among those identified in prickly pear samples were hydrox-
ycinnamic and benzoic acids, such as ferulic acid, gallic acid, caffeic acid and cinnamic acid
as well as and flavanones, such as hesperidin and naringin. These compounds had been
previously identified as the major compounds in orange samples (juice, wine and vine-
gar) [
40
]. Seven compounds presented similar concentrations in the three studied matrices,
Foods 2022,11, 303 16 of 23
such as gallic acid, hydroxy-tyrosol, tyrosol or ferulic acid. The rest of the phenolic com-
pounds studied showed significantly different contents when the matrix was transformed
from prickly pear juice into prickly pear vinegar. A few compounds (epigallocatechin,
syringic acid, protocatechualdehyde and cinnamic acid) increased their concentrations
gradually during the elaboration process of the vinegar. On the other hand, just two
compounds (ethyl gallate and p-hydroxybenzoic acid) decreased their concentration as
the juice was transformed into vinegar. Other authors have also reported decreases of
phenolic compounds over the production of pomegranate vinegars [
47
,
48
]. In our case,
hesperidin’s concentration decreased significantly during the alcoholic fermentation and
later on increased when the acetic fermentation was accomplished, whereas m-coumaric
acid disappeared as the transformation process from prickly pear juice into prickly pear
vinegar took place. Caffeic acid and vanillic acid were not detected in prickly pear juice
or wine, but they were present after the acetic fermentation process, and epigallocatechin
and cinnamic acid were not detected in the juice stage, but were identified in the following
stages. A possible hypothesis could be that these compounds were produced and/or
released during the fermentation processes. In fact, it has been previously reported that by
the selection of the bacteria strain employed in the fermentation process, some bioactive
components could be promoted to the final product [
15
]. Tyrosol was the main compound
found in all the samples and it was actually detected at a high concentration in juice, which
would explain its presence in the vinegar. This is not in agreement with the results from
certain previous studies on other fruit vinegars, where an increase in tyrosol content after
the alcoholic fermentation was reported [
33
,
41
]. With regard to gallic acid, a previous study
reported that it was the main phenolic compound at variable concentrations in a number of
varieties of prickly pear juice, except for the juice obtained from Tapona fruit, where no
gallic was detected. Syringic acid was the second most abundant phenolic compound in the
juices, although it was not identified in the juice from two of the studied varieties [
49
]. No
other investigations concerning the phenolic composition of prickly pear wine or vinegar
have been found in the literature.
In order to gain further insight into the evolution of the process, the polyphenolic
data obtained were also submitted to multivariate statistical study (principal component
analysis, PCA and cluster analysis, CA). Thus, four PCs were detected that were able to
explain 78.92% of the total variability according to the Kaiser criterion (eigenvalues > 1).
By attending to just the first two components, 61.99% of the variability could be explained.
Figure 4shows the plot, including the samples on the plane defined by PC1 and PC2.
As can be seen, PC1 was able to separate the three prickly pear matrices, whereas PC2
separated the juice samples from the wine and vinegar ones. The polyphenolic compounds
that contributed more and with a greater influence on PC1 were as follows: epigallocatechin,
tyrosol, syringic acid, protocatechualdehyde and caffeic acid, whereas PC2 was affected
mainly by ethyl gallate, m-coumaric acid, hesperidin and p-hydroxybenzoic acid. Regarding
the CA, the squared Euclidean distance as metric and the Ward method as the amalgamation
rule were employed to set up the clusters. As can be seen (Figure 5), three groups were
obtained as follows: juice and wine; vinegars produced at 30
◦
C and vinegars produced at
37 ◦C.
Table 5shows the results from the Analysis of Variance on the polyphenolic composi-
tion of prickly pear vinegars according to temperature and bacteria inoculum differences.
As can be seen, the concentration of polyphenolic compounds was significantly affected
by both fermentation temperature and acetic acid bacteria inoculum. Except for gallic
and vanillic acid, the concentration levels of all the polyphenolic compounds identified
in prickly pear vinegars were affected by fermentation temperature changes, so that tem-
perature was confirmed as the most important variable. Similar results, regarding other
analytes, had been obtained by Kim et al. when elaborating tomato vinegar [45].
Table 6includes the mean concentrations of the polyphenolic composition of the vine-
gars produced at 30
◦
C and at 37
◦
C and using the three types of bacteria inocula. It can be
seen that contrarily to the behavior exhibited by their volatile composition, the vinegar that
Foods 2022,11, 303 17 of 23
had been produced at 37
◦
C presented significantly higher concentrations of most phenolic
compounds, except for gallic acid, hydroxy-tyrosol, vanillic acid, quercetin, cinnamic acid
and p-hydroxybenzoic acid. It could, therefore, be said that, in terms of polyphenolic
composition and considering the beneficial antioxidant effect from polyphenols reported
by other studies on fruit vinegars, the vinegars produced at higher temperatures might
be healthier than those produced at 30
◦
C [
15
]. Regarding the type of bacteria inoculum
employed, no clear trend has been revealed in terms of a higher or lower concentration of
polyphenolic compounds associated with the particular bacteria inoculum used to produce
the vinegars (Table 6).
Foods 2022, 11, x FOR PEER REVIEW 18 of 25
varieties [49]. No other investigations concerning the phenolic composition of prickly pear
wine or vinegar have been found in the literature.
In order to gain further insight into the evolution of the process, the polyphenolic
data obtained were also submitted to multivariate statistical study (principal component
analysis, PCA and cluster analysis, CA). Thus, four PCs were detected that were able to
explain 78.92% of the total variability according to the Kaiser criterion (eigenvalues > 1).
By attending to just the first two components, 61.99% of the variability could be explained.
Figure 4 shows the plot, including the samples on the plane defined by PC1 and PC2.
Figure 4. PCA on phenolic compounds. Distribution of all the samples onto the plane defined by
the first two PCs. (A): original graph; (B): zoomed area. J: prickly pear juice; W: prickly pear wine;
A/G/M: prickly pear vinegar.
Figure 4.
PCA on phenolic compounds. Distribution of all the samples onto the plane defined by
the first two PCs. (
A
): original graph; (
B
): zoomed area. J: prickly pear juice; W: prickly pear wine;
A/G/M: prickly pear vinegar.
Foods 2022,11, 303 18 of 23
Foods 2022, 11, x FOR PEER REVIEW 19 of 25
As can be seen, PC1 was able to separate the three prickly pear matrices, whereas
PC2 separated the juice samples from the wine and vinegar ones. The polyphenolic com-
pounds that contributed more and with a greater influence on PC1 were as follows: epi-
gallocatechin, tyrosol, syringic acid, protocatechualdehyde and caffeic acid, whereas PC2
was affected mainly by ethyl gallate, m-coumaric acid, hesperidin and p-hydroxybenzoic
acid. Regarding the CA, the squared Euclidean distance as metric and the Ward method
as the amalgamation rule were employed to set up the clusters. As can be seen (Figure 5),
three groups were obtained as follows: juice and wine; vinegars produced at 30 °C and
vinegars produced at 37 °C.
Figure 5. Cluster analysis taking into consideration the composition of polyphenolic compounds of
the three prickly pear matrixes. J: prickly pear juice; W: prickly pear wine; A/G/M: prickly pear
vinegar.
Table 5 shows the results from the Analysis of Variance on the polyphenolic compo-
sition of prickly pear vinegars according to temperature and bacteria inoculum differ-
ences. As can be seen, the concentration of polyphenolic compounds was significantly
affected by both fermentation temperature and acetic acid bacteria inoculum. Except for
gallic and vanillic acid, the concentration levels of all the polyphenolic compounds iden-
tified in prickly pear vinegars were affected by fermentation temperature changes, so that
temperature was confirmed as the most important variable. Similar results, regarding
other analytes, had been obtained by Kim et al. when elaborating tomato vinegar [45].
Figure 5.
Cluster analysis taking into consideration the composition of polyphenolic compounds of the
three prickly pear matrixes. J: prickly pear juice; W: prickly pear wine; A/G/M: prickly pear vinegar.
Table 5.
Analysis of variance considering the effect of temperature (30
◦
C/37
◦
C) and bacteria
inoculum (Acetobacter (A)/Gluconobacter (G)/Mixture of bacteria (M)) on phenolic compounds of
prickly pear vinegar produced by surface culture.
Compounds Temperature (30 ◦C/37 ◦C) Bacteria Inoculum (A/G/M)
F Ratio p-Value F Ratio p-Value
Gallic acid 0.173 0.6836 5.95 0.0039 *
Hydroxy-tyrosol 115 0.0000 * 6.90 0.0017 *
Epigallocatechin 40.4 0.0000 * 2.90 0.0609
Tyrosol 19.2 0.0000 * 5.12 0.0080 *
Vanillic acid 1.74 0.1907 5.53 0.0056 *
Syringic acid 9.59 0.0027 * 4.46 0.0145 *
Hesperidin 7.60 0.0072 * 3.13 0.0491 *
Naringenin 107 0.0000 * 3.45 0.0363 *
Protocatechualdehyde 21.3 0.0000 * 4.00 0.0220 *
Caffeic acid 29.1 0.0000 * 0.583 0.5647
Ferulic acid 21.1 0.0000 * 1.24 0.2959
Quercetin 54.1 0.0000 * 1.08 0.3452
Cinnamic acid 21.2 0.0000 * 2.09 0.1299
p-Hydroxybenzoic acid 20.4 0.0000 * 1.59 0.2096
* Values are significant at p< 0.05.
These data on vinegar polyphenol content were submitted to a multivariate statistical
study (PCA) and three PCs were identified that were able to explain 73.28% of the total
variability (eigenvalues > 1). Figure 6illustrates which of the vinegar samples were located
on the plane defined by PC1 and PC2. Thus, it could be observed that these two components
were capable of separating all the vinegars produced at 30
◦
C from those fermented at 37
◦
C,
with 37
◦
C-elaborated vinegars located at the positive values of PC1, whereas the 30
◦
C-
elaborated vinegars were located at the negative value area of the same PC. The compounds
that contributed the most to the first principal component (PC1) were epigallocatechin,
tyrosol and protocatechualdehyde, while for PC2, gallic acid, hydroxy-tyrosol, vanillic acid
and p-hydroxybenzoic acid.
Foods 2022,11, 303 19 of 23
Table 6.
Mean concentrations (ppm) and standard deviations of phenolic compounds identified by
UPLC-DAD in prickly pear vinegar produced by surface culture method at 30
◦
C and 37
◦
C, and for
the three types of bacteria.
Compounds 30 ◦C 37 ◦CAcetobacter Gluconobacter Mixture
Mean ±SD Mean ±SD Mean ±SD Mean ±SD Mean ±SD
Gallic acid 1.76 ±0.604 1.69 ±0.156 1.78 ±0.410 b 1.43 ±0.527 a 2.10 ±0.650 b
Hydroxy-tyrosol 2.71 ±1.63 b ND a 2.31 ±1.36 a 4.05 ±1.74 b 4.34 ±2.94 b
Epigallocatechin 7.51 ±1.49 a 8.97 ±0.631 b 8.39 ±1.27 7.16 ±2.23 8.49 ±2.34
Tyrosol 52.8 ±12.1 a 60.3 ±4.30 b 57.9 ±9.26 b 47.8 ±14.8 a 53.3 ±7.37 ab
Vanillic acid 2.39 ±0.738 1.38 ±0.029 2.19 ±0.776 b ND a ND ab
Syringic acid 2.24 ±0.504 a 2.42 ±0.168 b 2.38 ±0.369 b 2.01 ±0.601 a 2.14 ±0.232 ab
Ethyl gallate 7.41 ±1.77 a 9.74 ±1.59 b 8.72 ±2.08 7.50 ±2.37 7.33 ±2.11
m-Coumaric acid 2.82 ±0.328 a 4.46 ±0.484 b 3.63 ±0.935 a 3.47 ±1.16 ab 4.04 ±0.676 b
Hesperidin 1.34 ±0.198 a 1.44 ±0.069 b 1.42 ±0.150 b 1.22 ±0.327 a 1.42 ±0.256 ab
Naringenin 1.01 ±0.082 a 1.11 ±0.065 b 1.06 ±0.083 0.992 ±0.261 1.09 ±0.168
Protocatechualdehyde 1.30 ±0.233 a 1.53 ±0.180 b 1.43 ±0.238 1.26 ±0.359 1.38 ±0.373
Caffeic acid 1.13 ±0.226 a 1.53 ±0.204 b 1.35 ±0.279 1.19 ±0.412 1.38 ±0.473
Ferulic acid 0.093 ±0.045 a 0.159 ±0.068 b 0.129 ±0.067 0.100 ±0.057 0.130 ±0.131
Quercetin 1.36 ±0.532 b 0.902 ±0.144 a 1.15 ±0.472 1.02 ±0.376 1.34 ±0.426
Cinnamic acid 1.76 ±0.604 1.69 ±0.156 1.78 ±0.410 b 1.43 ±0.527 a 2.10 ±0.650 b
p-Hydroxybenzoic acid 2.71 ±1.63 b ND a 2.31 ±1.36 a 4.05 ±1.74 b 4.34 ±2.94 b
For each variable (temperature and bacteria) different letters in the same row indicate significant differences
according to Tukey’s test (p< 0.05); ND: Not detected.
Foods 2022, 11, x FOR PEER REVIEW 21 of 25
These data on vinegar polyphenol content were submitted to a multivariate statistical
study (PCA) and three PCs were identified that were able to explain 73.28% of the total
variability (eigenvalues > 1). Figure 6 illustrates which of the vinegar samples were located
on the plane defined by PC1 and PC2. Thus, it could be observed that these two compo-
nents were capable of separating all the vinegars produced at 30 °C from those fermented
at 37 °C, with 37 °C-elaborated vinegars located at the positive values of PC1, whereas the
30 °C-elaborated vinegars were located at the negative value area of the same PC. The
compounds that contributed the most to the first principal component (PC1) were epigal-
locatechin, tyrosol and protocatechualdehyde, while for PC2, gallic acid, hydroxy-tyrosol,
vanillic acid and p-hydroxybenzoic acid.
Figure 6. PCA on phenolic compounds. Distribution of all the vinegar samples onto the plane de-
fined by the first two PCs. A 30/A 37: Acetobacter at 30 °C and 37 °C, G 30/G 37: Gluconobacter at 30
°C and 37 °C, M 30/M 37: Mixture of bacteria at 30 °C and 37 °C.
3.3.2. Antioxidant Activity
The antioxidant activity of prickly pear samples (juice, wine and vinegars) was de-
termined by means of DPPH radical scavenging (expressed as EC
20
). This is a fast, simple,
economical and widely used method to measure the overall antioxidant capacity and the
free radical scavenging activity of fruits and vegetable juices. This method is carried out
in a mixture methanol/water, which facilitates the extraction of antioxidant compounds
from the sample. Moreover, DPPH is allowed to react with the whole sample and suffi-
cient time given in the method allows DPPH to react slowly even with weak antioxidants;
however, this method is limited because DPPH radical interacts with other radicals and
some problems of linearity can be found with different ratios of antioxidant/DPPH. In
addition, DPPH is sensitive to some Lewis bases and solvent types, as well as oxygen, and
it can only be soluble in organic solvents. Another drawback is that the absorbance of
DPPH in methanol and acetone decreases under light [50]. In spite of these drawbacks,
this methodology is nowadays commonly employed thanks to the above-mentioned ad-
vantages.
Depending on the matrix, the results revealed that there was a clear difference with
respect to the antioxidant activity corresponding to juice, wine or vinegars (0.618 ± 0.000
mg/mL, 0.456 ± 0.035 mg/mL, 1.459 ± 0.055 mg/mL, respectively). Although there is no
clear evidence that in vitro antioxidant activity values have relation to a biological signif-
Figure 6.
PCA on phenolic compounds. Distribution of all the vinegar samples onto the plane defined
by the first two PCs. A 30/A 37: Acetobacter at 30
◦
C and 37
◦
C, G 30/G 37: Gluconobacter at 30
◦
C
and 37 ◦C, M 30/M 37: Mixture of bacteria at 30 ◦C and 37 ◦C.
3.3.2. Antioxidant Activity
The antioxidant activity of prickly pear samples (juice, wine and vinegars) was deter-
mined by means of DPPH radical scavenging (expressed as EC
20
). This is a fast, simple,
economical and widely used method to measure the overall antioxidant capacity and the
free radical scavenging activity of fruits and vegetable juices. This method is carried out in
a mixture methanol/water, which facilitates the extraction of antioxidant compounds from
Foods 2022,11, 303 20 of 23
the sample. Moreover, DPPH is allowed to react with the whole sample and sufficient time
given in the method allows DPPH to react slowly even with weak antioxidants; however,
this method is limited because DPPH radical interacts with other radicals and some prob-
lems of linearity can be found with different ratios of antioxidant/DPPH. In addition, DPPH
is sensitive to some Lewis bases and solvent types, as well as oxygen, and it can only be
soluble in organic solvents. Another drawback is that the absorbance of DPPH in methanol
and acetone decreases under light [
50
]. In spite of these drawbacks, this methodology is
nowadays commonly employed thanks to the above-mentioned advantages.
Depending on the matrix, the results revealed that there was a clear difference with
respect to the antioxidant activity corresponding to juice, wine or vinegars (0.618
±
0.000
mg/mL, 0.456
±
0.035 mg/mL, 1.459
±
0.055 mg/mL, respectively). Although there is
no clear evidence that
in vitro
antioxidant activity values have relation to a biological
significance after the consumption of any food, it could provide an initial idea about their
possible healthy character. The DPPH antioxidant scavenging capacity of prickly pear juice
from Moroccan O. ficus indica in our study was similar to that registered for Tunisian O. ficus
indica pulp [
51
]. As can be seen, the highest antiradical activity was exhibited by the prickly
pear wine with the lowest EC
20
values, which might be explained by the solubility of the
polymerized polyphenols in ethanol, that resulted in antioxidant compounds concentration
increments as the content of ethanol increased over the alcoholic fermentation. The results
obtained in our study are in contradiction with those obtained by Kongkiattikajorn [
52
],
who reported that the total antioxidant activity of Roselle vinegar was significantly higher
than that of Roselle juice and wine because of a greater number of anthocyanins in the
vinegar in relation to that found in the wine and juice. A previous study on the evolution
of the antioxidant capacity of fermenting persimmon juice determined by DPPH assays,
showed that the antioxidant capacity went up during the alcoholic fermentation and
acetification, which is in line with the presence of a greater amount of flavan-3-ols and
condensed tannins [53].
Regarding the antioxidant activity of vinegars produced by inoculating different
bacteria genus, the data revealed that the vinegars produced by the bacteria mixture
from Sherry vinegar displayed a greater activity (0.833
±
0.0440 mg/mL) than those
produced by means of either Gluconobacter (1.018
±
0.030 mg/mL) or Acetobacter genus
(
1.636 ±0.061 mg/mL
). This may be explained by a combination of a greater amount of
certain bioactive compounds such as hydroxy-tyrosol or p-hydroxybenzoic acid in the
vinegar produced by the mixture of bacteria found in Sherry vinegar lees, compared
to those obtained by Gluconobacter and Acetobacter, in decreasing order (Table 6). These
two phenolic compounds have been previously related to the high antioxidant activity of
other vegetal matrices such as Jasminum species [
54
]. In addition, hydroxy-tyrosol is also
considered the main responsible for the bioactivity of olives and olive pits [55].
4. Conclusions
Prickly pear vinegar has been produced by the surface culture at different temperatures
and with different AAB inocula. The yields from fermentations at 30
◦
C ranged between
67.47% and 86.89%, whereas the yields at 37
◦
C did not reach over 21.83% in any case.
Eighty-five separate volatile compounds from different families and sixteen polyphenolic
compounds have been identified in the vinegars. By observing the effect of the acetification
temperature on volatile compounds, it was concluded that at the highest temperature tested
(37
◦
C), the concentration of these compounds decreased; however, the highest content
of polyphenolic compounds was detected when the vinegars were produced at 37
◦
C.
Further, a greater antioxidant activity was exhibited by prickly pear wine than by juice or
vinegars. The vinegar produced by a mixture of AAB from Sherry vinegar displayed the
highest antiradical activity compared to those corresponding to the vinegars elaborated
using other AAB inocula. It can be concluded that changes in the temperature levels during
the fermentation of prickly pear to produce vinegar can have a significant impact on the
polyphenolic and volatile composition of the final vinegars, and although their volatile
Foods 2022,11, 303 21 of 23
content decreases when fermented at higher temperature, partial fermentations could be
carried out at 37
◦
C as long as thermotolerant bacteria are employed, so that the final
vinegar would be richer in polyphenols.
Author Contributions:
Conceptualization, E.D.-G. and R.C.; methodology, E.D.-G.; software, R.C.;
validation, Y.C.-J.; formal analysis, I.E.-s. and Y.C.-J.; investigation, I.E.-s.; resources, R.C.; data
curation, I.E.-s.; writing—original draft preparation, I.E.-s.; writing—review and editing, E.D.-G.;
visualization, R.Z.; supervision, E.D.-G.; project administration, R.Z.; funding acquisition, R.C. All
authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement: Data is contained within the article.
Conflicts of Interest: The authors declare no conflict of interest.
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